CA2760534A1 - Compositions and methods for generating an immune response utilizing alphavirus-based vector systems - Google Patents

Abstract

Methods are provided for generating immune responses utilizing alphavirus-based vector systems.

Description

COMPOSITIONS AND METHODS FOR GENERATING AN IMMUNE RESPONSE
UTILIZING ALPHAVIRUS-BASED VECTOR SYSTEMS

This application is a divisional application of Canadian Patent Application Number 2,373,300, filed on April 14, 2000.

TECHNICAL FIELD
The present invention relates generally to gene-based vaccines and therapeutics, and more specifically, to compositions and methods that increase the efficiency of alphavirus-based vector systems used for such vaccines and therapeutics.
BACKGROUND OF THE INVENTION
Alphaviruses comprise a group of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. These viruses are distributed worldwide, and persist in nature through a mosquito to vertebrate cycle.
Birds, rodents, horses, primates, and humans are among the identified alphavirus vertebrate reservoir/hosts.
Twenty-six known viruses and virus subtypes have been classified within the alphavirus genus utilizing the hemagglutination inhibition (HI) assay. This assay segregates the 26 alphaviruses into three major complexes: the Venezuelan equine encephalitis (VEE) complex, the Semliki Forest (SF) complex, and the western equine encephalitis (WEE) complex. In addition, four other viruses. eastern equine encephalitis (EEE), Barmah Forest. Middelburg, and Ndumu, receive individual classification based on the HI serological assay.
Members of the alphavirus genus also are further classified into one of two groups, according to the clinical symptoms they exhibit as a result of infection in humans. The first group is alphaviruses associated primarily with encephalitis, and the second group is alphaviruses associated primarily with fever. rash, and polyarthritis.
Included in the first group are the VEE and WEE complexes. and EEE. In general, infection with this group can result in permanent sequelae. including death.
In the second group is the SF complex, comprised of the individual alphaviruses Semliki Forest, Sindbis, Ross River, Chikungunya, O'nyong-nyong, and Mayaro. Although serious epidemics have been reported, infection by viruses of this group is generally self-limiting, without permanent sequelae.

Sindbis virus is the prototype member of the .41pfiavirus genus of the Togaviridae family. Its replication strategy is well characterized and serves as a model for other alphaviruses (Strauss and Strauss, Aficrobio. Rev. 58:491-562, 1994). The genome of Sindbis virus (like other alphaviruses) is an approximately 12 kb single-stranded, positive-sense RNA molecule that is capped and polyadenylated.
Genome RNA is contained within a virus-encoded capsid protein shell which is, in turn, surrounded by a host-derived lipid envelope from which two viral-specific glycoproteins, El and E2, protrude as spikes from the virion surface. Certain alphaviruses (e.g., SF) also maintain an additional protein, E3, which is a cleavage product from the E2 precursor protein, PE2.
After virus particle absorption to target cells, penetration, and uncoating of the nucleocapsid to release viral genomic RNA into the cytoplasm, the replication process is initiated by translation of four nonstructural replicase proteins (nsP]-nsP4) from the 5' two-thirds of the viral genome. The four nsPs are translated as one of two polyproteins (nsPl23 or nsP1234), and processed post-translationally into mature monomeric proteins by an active protease in the C-terminal domain of nsP2.
Both of the nonstructural polyproteins and their derived monomeric units may participate in the RNA replication process, which involves nsP binding to the conserved nucleotide sequence elements (CSEs) present at the 5' and 3' ends, and an internal subgenomic junction region promoter.

The positive strand genome RNA serves as template for the nsP-catalyzed svnthesis of a full-length complementary negative strand RNA.
Synthesis of the negative strand RNA is catalyzed by binding of a nsP complex to the 3' terminal CSE of the positive strand genome RNA. The negative strand, in turn, serves as template for the synthesis of additional positive strand genome RNA, as well as an abundant subgenomic RNA, initiated internally at the junction region promoter.
Synthesis of additional positive strand genome RNA occurs after binding of a nsP
complex to the 3' terminal CSE of the complementary negative strand genome-length RNA template. Synthesis of the subgenomic mRNA from the negative-strand RNA
template is initiated from the junction region promoter. Thus, the 5' end and junction region CSEs of the positive strand genome RNA are functional only after being transcribed into the negative strand RNA complement (i.e., the 5' end CSE is functional when it is the 3' end of the genomic negative stranded complement).
Alphavirus structural proteins (sPs) are translated from the subgenomic RNA, which represents the 3' one-third of the genome, and like the nsPs, are processed post-translationally into the individual proteins (see Figure 1). Translation of this subgenomic mRNA produces a single polyprotein consisting of the structural proteins capsid (C) glycoprotein E2 and glycoprotein El, plus the corresponding leader/signal sequences (E3, 6k) for glycoprotein insertion into the endoplasmic reticulum.
The structural gene polyprotein is processed into the mature protein species by a combination of viral (capsid autoprotease) and cellular proteases (e.g., signal peptidase).
Alphavirus structural proteins are produced at very high levels due to the abundance of subgenomic mRNA transcribed. as well as the presence of a translational enhancer element (Frolov and Schlesinger, J. Virol. 68:8111-8117, 1994; Sjoberg et al., Bio/Technol. 12:1127-1131, 1994) within the mRNA, located in the capsid gene coding sequence. Because all structural proteins are synthesized at equimolar ratios, as part of the polyprotein, the translation enhancer element exerts its effect equally on each of the genes.
Several members of the Alphavirus genus are being developed as expression vectors, including, for example, Sindbis virus (Xiong et al., Science 243:1188-1191, 1989; Halm et at., Proc. Nall. Acad. Sci. USA 89:2679-2683, 1992;
Dubensky et al., J. Virol. 70:508-519, 1996), Semliki Forest virus (Liljestrom, Bio/Technologv 9:1356-1361, 1991), and Venezuelan equine encephalitis virus (Pushko et at., Virology 239:389-401, 1997). The general strategy for construction of alphavirus-based expression vectors has been to substitute the viral structural protein genes with a heterologous gene, maintaining transcriptional control via the highly active subgenomic RNA promoter. RNA vectors having this configuration are self-amplifying, and are termed RNA "replicons" and may be synthesized iii vitro from cDNA using a bacteriophage promoter (Xiong et al., ibid; Liljestrom et al., ibid; Pusliko et at, ibid), or generated in vivo directly from DNA when linked to a eukaryotic promoter (Dubensky et al., ibid; US Patent 5,814,482). Because the vector replicons do not express the alphavirus structural proteins necessary for packaging into recombinant alphavirus particles, these proteins must be provided in trans. One alphavirus, Venezuelan equine encephalitis virus, and its derived recombinant vector particles have been shown to be lymphotropic and infect murine dendritic cells (Caley et al., J Virol.
71:3031-3038, 1997; MacDonald et al., J Virol. 74:914-22, 2000). However, no alphavirus or alphavirus variant was demonstrated to infect human dendritic cells, macrophages or antigen presenting cells.
The present invention discloses novel compositions and methods for generating an enhanced immune response utilizing alphavirus-based vector systems, and further, provides other related advantages.

SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and methods for generating an enhanced immune response utilizing alphavirus-based vector systems.
Within one aspect of the present invention, isolated alphaviruses and recombinant alphavirus particles are provided which infect human dendritic cells. (with the proviso that the alphavirus is not ATCC # VR-2526 or said alphavirus particle is not generated in total from ATCC # VR-2526). Within another aspect. isolated alphaviruses and recombinant alphavirus particles are provided which infect non-human dendritic cells (with the proviso that the alphavirus is not a Venezuelan equine encephalitis virus or ATCC # VR-2526, or, that the recombinant alphavirus particle is not derived in total from Venezuelan equine encephalitis virus or ATCC # VR-2526). Within vct another aspect- isolated alphaviruscs and recombinant alphavirus particles are provided which infect human macrophages. \\'ithin related aspects. isolated alphaviruses and recombinant alphavirus particles are provided which in(cet human antigen presenting cells. (with the proviso that the alphavirus is not ATCC VR-2526 or said alphavirus panicle is not 1 cneratcd in total from ATCC T VR-2526).
Within certain embodiments of the above, the alphavirus or recombinant alphavirus particle has an amino acid substitution in the E2 glycoprotein as compared to wild-type, for example, at residue 158, 169, 160, 161, or, 162. Within preferred embodiments, the amino acid substitution is at E2 residue 160. Within other embodiments, the alphavirus has an amino acid deletion or insertion in the E2 glycoprotein. Within further ct'nbodiments, the alphavirus is a Semliki Forest virus, a Ross River virus, a Venezuelan equine encephalitis virus, a Sindbis virus, or ATCC No.
VR-2643. Also provided are nucleic acid molecules which encode the above described aiphaviruses (such as, for example, as provided in Figures 2B and 2C).
Within other aspects of the present invention alphavirus structural protein expression cassettes are provided, comprising a promoter operably linked to a nucleic acid sequence encoding one or more alphavirus structural proteins from an alphavirus as described above. Within a related aspect, alphavirus structural protein expression cassettes are also provided comprising a promoter operably linked to a nucleic acid sequence encoding alphavirus structural proteins, wherein said nucleic acid sequence comprises a sequence encoding glycoprotein E2, and wherein said sequence encodes a mutation (e.g., a substitution, deletion, or insertion, as compared to wild-type) in the E2 glycoprotein. Within various embodiments, the mutation is a substitution at E2 residue 158, 159, 160, 161, or 162. Within preferred embodiments, the mutation confers to an alphavirus or recombinant alphavirus particle packaged with said E2 glycoprotein the ability to infect human dendritic cells. Within related aspects alphavirus packaging cell lines are provided comprising a host cell and an alphavirus structural protein expression cassette as described above, as well as an alphavirus producer cell line comprising such packaging cells and a vector selected from the group consisting of an alphavirus RNA vector replicon. alphavirus vector construct, and a eukaryotic layered vector initiation system. Further, recombinant alphavirus particles also are provided which can be produced from the above packaging or producer cell lines.
Within yet other aspects of the present invention, methods are provided for introducing a selected sequence into a cell, comprising the step of infecting or otherwise introducing into a cell a recombinant alphavirus particle as described above, such that the selected sequence is introduced into the cell. A wide variety of selected sequences may be introduced into a cell or cells, including for example, heterologous sequences such as proteins (e.g., peptides, antigens, antibodies), and non-protein sequences such as ribozymes. In addition, the recombinant alphavirus particles may be designed to express one or more sequences (e.g., an immune enhancer, cytokine, chemokine, for example, IL-2, IL-l0, IL-l2, gamma interferon. GM-CSF, MIP3a.
MIP3J3, molecules with related function). Further, the recombinant alphavirus particles may be administered utilizing ex vivo or in vivo techniques. Further, within certain embodiments the recombinant alphavirus particles may be utilized with a wide variety of cells, cell populations, or tissues, including for example, a population of cells containing dendritic cells. Within yet further embodiments, the recombinant alphavirus particles may be introduced as a composition, e.g., with enhancers such as cytokines or chemokines (e.g., IL-2, IL-10, IL-12, gamma interferon, GM-CSF), other vectors, or adjuvants.
Within yet further aspects of the invention, alphavirus_ vector constructs are provided comprising (a) a 5' promoter which initiates synthesis of viral RNA
in vitro from cDNA, (b) a S' sequence which initiates transcription of alphavirus RNA, (c) a nucleic acid molecule which operably encodes all four aiphaviraI
nonstructural proteins, (d) an alphavirus RNA polymerase recognition sequence, and (e) a 3'polyadenylate tract, wherein the nucleic acid sequence which operably encodes all four alphaviral nonstructural proteins contains a mutation in at least one nonstructural protein selected from the group consisting of a mutation in nsP 1 residues 346, 441. 473, nsP2 residues 438. 622, 634, 715, nsP3 residues, 417, 456, 505. and nsP4 residue 266.

Within yet another aspects of the present invention, eukaryotic layered vector initiation systems are provided, comprising a 5' promoter capable of initiating in vivo the 5' synthesis of alphavirus RNA from cDNA, a sequence which initiates transcription of alphavirus RNA following the 5' promoter, a nucleic acid molecule which operably encodes all four alphaviral nonstructural proteins, an alphavirus RNA
polymerase recognition sequence, and a 3' polyadenylate tract, wherein the nucleic acid sequence which operably encodes all four alphaviral nonstructural proteins contains a mutation in at least one nonstructural protein selected from the group consisting of a mutation in nsPl residues 346, 441, 473, nsP2 residues 438, 622, 634, 715, nsP3 residues, 417, 456, 505, and nsP4 residue 266, as compared to wild-type.
Within further embodiments alphavirus RNA vector replicons capable of translation in a eukaryotic system are provided, comprising a 5' sequence which initiates transcription of alphavirus RNA, a nucleic acid molecule which operably encodes all four alphaviral nonstructural proteins, an alphavirus RNA polymerase recognition sequence and a 3' polyadenylate tract, wherein the nucleic acid sequence which operably encodes all four alphaviral nonstructural proteins contains a mutation in at least one nonstructural protein selected from the group consisting of a mutation in nsPl residues 346, 441, 473, nsP2 residues 438, 622, 634, 715, nsP3 residues, 417, 456, 505, and nsP4 residue 266, as compared to wild-type.
Within another aspect of the invention, methods are provided for generating an immune response in a warm-blood animal, comprising the step of administering to a warm-blooded animal any of the recombinant alphavirus particles described above that further comprise a heterologous sequence encoding an antigen from a virus, bacteria, fungus, parasite or cancerous cell.
Within a further aspect of the invention, methods are providing for generating an immune response within a warm-blooded animal. comprising the step of administering to a warm blooded animal one or more selected antigens by a first method (e.g., alphavirus vector particles, eukaryotic layered vector initiation system. DNA, protein), followed by administering to the same animal the same or similar antigen by a second method (e.g., alphavirus vector particles. eukaryotic layered vector initiation system, DNA, protein), wherein the first and second methods are different, with the proviso that either the first method or the second method or both methods is selected from the group consisting of alphavirus vector particles, RNA vector replicons or eukaryotic layered vector initiation systems. Within various embodiments, the immune response may be generated to treat a disease, and/or to prevent a disease (e.g., as a vaccine).
Within another aspect of the invention, non-lymphotropic alphaviruses are provided, wherein the alphaviruses are capable of infecting dendritic cells (DC
tropic). In one embodiment the dendritic cells are immature dendritic cells (e.g., CD 1 a+, CD86dim, CD83"). In another embodiment, the alphaviruses are selected from the group consisting of Sindbis virus. Semliki Forest virus, and Ross River virus. In yet another embodiment, the alphavirus is a Sindbis virus that has an amino acid encoded at residue 160 of glycoprotein E2 that is not glutamic acid. Within a further embodiment, the alphavirus is not a Venezuelan equine encephalitis virus or Sindbis virus CMCC
#4639 (deposited with the ATCC on April 2, 1996: VR-2526)_ Within a further embodiment the alphavirus has the nucleic acid sequence set forth in Figure 2B, or, a nucleic acid sequence which but for the redundancy of the genetic code encodes the same amino acid sequence. Within other aspects of the invention, non-lymphotropic alphaviruses are also provided, for example, as disclosed in Figure 2C, or, a nucleic acid sequence which but for the redundancy of the genetic code encodes the same amino acid sequence. Also provided are aiphavirus structural protein expression cassettes, RNA vector replicons, alphavirus vector particles, and eukaryotic layered vector initiation systems which are obtained or derived from, at least in part, one or more of the above-noted alphaviruses.
Within another aspect of the invention, alphavirus vector particles are provided, wherein the alphavirus vector particles are capable of infecting human dendritic cells with an efficiency of infection (percentage infected) greater than their efficiency for infecting murine dendritic cells. In one embodiment, the alphavirus vector particles can infect human dendritic cells at a 50%, 100%, 200%. or greater efficiency than their efficiency for infecting murine dendritic cells. In another embodiment the dendritic cells are immature human dendritic cells (e.g., CDla+, CD86dim. CD83-). In yet another embodiment, the alphavirus vector particles are derived from an alphaviruses selected from the group consisting of Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. Inyet another embodiment, the alphavirus vector particles are from Sindbis virus that has an amino acid encoded at residue 160 of glycoprotein E2 that is not glutamic acid. Within a further embodiment, the alphavirus vector particles are not derived from Sindbis virus CMCC #4639 (deposited with the ATCC on April 2. 1996: VR-2526). Within a further embodiment, the alphavirus vector particles are derived from the nucleic acid sequence set forth in Figure 2B, or, a nucleic acid sequence which but for the redundancy of the genetic code encodes the sag1e amino acid sequence. Also provided are alphavirus structural protein expression cassettes, RNA vector replicons, and Eukaryotic Layered Vector Initiation Systems which are obtained or derived, at least in part, from one or more of the above-noted alphaviruses.

Within other aspects of the present invention, expression cassettes are provided comprising a promoter (e.g., a bacteriophage promoter for in vitro use, or an RNA pol II promoter for in vivo use) operably linked to a full-length cDNA
clone of a DC tropic alphavirus from above, such that transcription of the full-length cDNA clone yields an RNA that initiates a productive viral infection when introduced into the cytoplasm of a susceptible cell. Also provided are alphavirus vector constructs, RNA
replicons, cDNA vector construct, ELVIS, and the like which are derived from the above DC tropic alphaviruses. Within certain embodiments, vectors are provided comprising a 5'-promoter (bacteriophage or RNApoIII) operably linked to the alphavirus vector cDNA sequence, subgenomic junction region promoter, heterologous gene to be expressed, and a polyadenylate tract.
A wide variety of antigens may be expressed from the alphavirus-based vector systems, including for example, antigens or peptides from a pathogenic agent (e.g., a cancerous cell, virus, bacteria, fungi, or parasite).
Within other aspects of the invention, a method of introducing and expressing a heterologous sequence in human dendritic cells in vivo or in vitro (e.g., for use in biological assays) is provided, comprising: infecting a population of human cells containing dendritic cells and/or dendritic cell precursors with a recombinant alphavirus vector particle according to the present invention for a time and under conditions necessary for intracellular expression of the replicon encoded heterologous gene within the dendritic cell, said alphavirus vector particle containing the heterologous sequence, with the proviso that said alphavirus vector particle is not derived entirely from Sindbis virus CMCC #4639 (deposited with the ATCC on April 2, 1996: VR-2526). However, such particle may contain a portion of CMCC #4639. Within certain embodiments, the alphavirus vector particle is from at least a portion of a Sindbis virus, Semliki Forest virus, Venezuelan equine encephalitis virus or Ross River virus.
Within another aspect of the invention, a method of introducing and expressing a heterologous sequence in human dendritic cells and also inducing activation and/or maturation of said dendritic cells is provided, comprising:
infecting a population of human cells containing dendritic cells and/or dendritic cell precursors with a recombinant alphavirus vector particle according to the present invention for a time and under conditions necessary for intracellular expression of the replicon encoded heterologous gene and activation and/or maturation of the dendritic cells, said alphavirus vector particle containing the heterologous sequence. Also provided are alphavirus vector particles comprising a vector replicon (as described in more detail below) and structural proteins necessary for particle formation. with the proviso that at least one structural protein of the vector particle is from a DC tropic alphavirus and said alphavirus vector particle is not derived entirely from Sindbis virus CMCC
#4639 (deposited with the ATCC on April 2, 1996: VR-2526; however, such particles may contain a portion of CMCC #4639). Within certain preferred embodiments, the vector replicon further comprises a gene encoding an antigen from a pathogenic agent.
Within other aspects of the invention, a method of introducing and expressing a heterologous sequence in dendritic cells is provided, comprising the step of infecting a population of cells containing dendritic cells and/or dendritic cell precursors with a recombinant alphavirus vector particle, the alphavirus vector particle containing the heterologous sequence and with the proviso that the alphavirus vector particle is not II
derived entirely from Venezuelan equine encephalitis virus or Sindbis virus CMCC
#4639 (deposited with the ATCC on April 2, 1996: VR-2526 (however, such particle may contain a portion of VEE or CMCC #4639)). Within certain embodiments, the alphavirus vector particle is from at least a portion of a Sindbis virus, Semliki Forest virus, or Ross River virus.
In further embodiments of the invention, the dendritic cells are immature dendritic cells. In another embodiment of the invention, said method of introducing and expressing a heterologous sequence in dendritic cells is carried out in vitro.
Such dendritic cells may be removed from peripheral blood by apheresis or other means, and/or separated or enriched from bone marrow, or from cultured and/or expanded or differentiated hematopoetic cells (see, e.g., U.S. Patent Nos. 4,927,750, 5,643,786, 5,646,004, 5,648,219, 5,648,248, 5,663,051, 5,788,963, 5,811,297, 5,851,756, 5,866,115 and 5,871,728; see also the Examples). In yet another embodiment of the invention, the method of introducing and expressing a heterologous sequence in dendritic cells is carried out in vivo, in a warm-blooded animal (e.g., by subcutaneous, intradermal, intramuscular, intranasal, intravenous injection).
Within other aspects of the invention. a method of introducing and expressing a heterologous sequence in human macrophages or antigen presenting cells in vivo or in vitro (e.g., for use in biological assays) is provided.
comprising: infecting a population of cells containint human macrophages or antigen presenting cells with a recombinant alphavirus vector particle according to the present invention for a Lime and under conditions necessary for intracellular expression of the rcplicon encoded heterologots gene within the dendritic cell. said alphavirus vector particle containing the heterologous sequence. with the proviso that said alphavirus vector particle is not derived entirely from Sindhis virus CMCC 44639 (deposited with the .ACC on April 2. 1996: VR-2526 (however. such particle may contain a portion of C\ICC
x=4639)).
Within certain embodiments. the alphavirus vector particle is from at least a portion of a Sindbis virus, Semliki Forest virus. Venezuelan equine encephalitis virus or Ross River Virus.

Within other aspects. methods are provided for stimulating an immune response within a warm-blooded animal, comprising the general step of administering to a warm-blooded animal alphavirus vector particles described herein that infect dendritic cells and express an antigen or a portion thereof from a pathogenic agent.
Within one embodiment, the alphavirus vector particles are not derived entirely from Venezuelan equine encephalitis virus or Sindbis virus CMCC #4639 (deposited with the ATCC April 2, 1996: VR-2526 (however, such particles may contain a portion of VEE
or CMCC #4639)). Within certain embodiments, the alphavirus vector particle is from at least a portion of a Sindbis virus, Semliki Forest virus, or Ross River virus.
Within other aspects, methods are provided for stimulating an antigen-specific Tcell response. comprising the steps of infecting dendritic cells with alphavirus vector particles described herein that express an antigen from a pathogenic agent. and allowing presentation of said antigen by dendritic cells to T cells. such that said T cells are stimulated in an antigen-specific manner.
Within a further aspect of the present invention, isolated nucleic acid molecules encoding a Sindbis virus E2 glycoprotein gene are provided, wherein said nucleic acid molecule has an amino acid substitution or deletion for the wild-type glutarnic acid at codon 160, and with the proviso that said glycoprotein gene is not obtained from VEE or CMCC #4639. Within one embodiment. a glycine residue is substituted at codon 160. Also provided are structural protein expression cassettes comprising such nucleic acid molecules, and alphavirus vector particles containing the above noted E2 structural protein.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain procedures or compositions (e.g., plasmids, etc.) BRIEF DESCRIPTION OF THE DRAWINGS

Figure I is a schematic illustration of one representative flow chart of a human dendritic cell adaptation strategy for alphaviruses.
Figure 2A describes a general cloning strategy for the genome of a human dendritic cell adapted alphavirus (e.g., Sindbis virus) variant.

Figure 2B is the nucleotide sequence of SinDCChiron virus Figure 2C is the nucleotide sequence of SinChironLP virus Figure 3 shows Sindbis-GFP vector infection of human dendritic cells.
Figure 4 shows increased efficiency of human DC infection by a SIN-GFP vector with SinDCChiron-derived envelope glycoproteins, as compared to glycoproteins from another DC variant of Sindbis virus.
Figure 5 shows that SIN-GFP vectors with DC adapted glycoproteins specifically target immature human dendritic cells as compared to mature DCs.
Figure 6 shows laser cytometric analysis of DC variant SIN-GFP vector infected cells trafficking to the lymph node after administration to mice Figure 7 shows microscopy of DC variant SIN-GFP vector infected cells that have trafficked to the lymph node after administration to mice Figure 8 is a graph showing that alphaviruses (e.g., SFV or SIN-LP) are capable of infecting murine DC, but not human DC unless they are first modified or adapted for the efficient infection of such human DC (see for example SIN
DC+).
Figure 9 is a graph showing alphavirus (e.g., SIN) vector induced maturation and activation of human DC following transduction in vitro and murine DC
following transduction in vivo.
Figure 10 shows the utility of human DC-adapted alphavirus vectors for in vitro assays that measure antigen presentation by transduced DC and stimulation of immune cells (e.g., T cells).
Figure 11 is a graph comparing immune induction using recombinant alphavirus particles containing either the new SINCR replicon or a wild-type SINBV
replicon.

Figure 12 is a graph that shows CTL enhancement by selected primeiboost vaccine strategies.

DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
Prior to setting forth the invention, it may be helpful to an understanding thereof to first set forth definitions of certain terms that will be used hereinafter.

An "isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated in the genomic DNA of an organism, or, in the case of the virus, not contained within the genome of a wild-type virus. One example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule, or, a nucleic acid molecule that is produced by recombinant (e.g., PCR) techniques.

"Genomic RNA" refers to an RNA that contains all of the genetic information required to direct its own amplification or self-replication in vivo, within a target cell. An alphavirus-derived genomic RNA molecule should contain the following ordered elements: 5' viral or defective-interfering RNA sequence(s) required in cis for replication, sequences which, when expressed, code for biologically active alphavirus nonstructural proteins (e.g., nsPI, nsP2, nsP3, nsP4), 3' viral sequences required in cis for replication. and a polyadenylate tract. The alphavirus-derived genomic RNA
vector replicon also may contain a viral subgenomic "junction region" promoter and sequences which, when expressed, code for biologically active alphavirus structural proteins (e.g., C, E3. E2, 6K, E 1). Generally, the term genomic RNA refers to a molecule of positive polarity, or "message" sense, and the genomic RNA may be of length different from that of any known, naturally-occurring alphavirus.

"Aiphavirus vector construct" refers to an assembly which is capable of directing the expression of a sequence or gene of interest. Such vector constructs are comprised of a 5' sequence which is capable of initiating transcription of an alphavirus RNA (also referred to as 5' CSE, in background), as well as sequences which, when expressed. code for biologically active alphavirus nonstructural proteins (e.g., nsPl.

nsP2, nsP3, nsP4), an alphavirus RNA polymerase recognition sequence (also referred to as 3' CSE, in background), and a polyadenylate tract. In addition, the vector construct may include a viral subgenomic `junction region" promoter, sequences from one or more structural protein genes or portions thereof, extraneous nucleic acid molecule(s) which are of a size sufficient to allow production of viable virus, a 5' promoter which is capable of initiating the synthesis of viral RNA from cDNA
in vitro or in vivo, a heterologous sequence to be expressed, and one or more restriction sites for insertion of heterologous sequences.
"Alphavirus RNA vector replicon", "RNA vector replicon" and "replicon" refers to an RNA molecule which is capable of directing its own amplification or self-replicationr in vivo, within a target cell. An alphavirus-derived RNA vector replicon should contain the following ordered elements: 5' viral sequences required in cis for replication (also referred to as 5' CSE, in background), sequences which, when expressed, code for biologically active alphavirus nonstructural proteins (e.g., nsP 1, nsP2, nsP3, nsP4), 3' viral sequences required in cis for replication (also referred to as 3' CSE, in background), and a polyadenylate tract. The alphavirus-derived RNA vector replicon also may contain a viral subgenomic "junction region"
promoter, sequences from one or more structural protein genes or portions thereof, extraneous nucleic acid molecule(s) which are of a size sufficient to allow production of viable virus, as well as heterologous sequence(s) to be expressed.
"Recombinant Alphavirus Particle, Alphavirus Vector Particle" refers to a virion-like structural unit containing an alphavirus RNA vector replicon.
Generally, a recombinant alphavirus particle comprises one or more alphavirus structural proteins, a lipid envelope and an RNA vector replicon. Preferably, the recombinant alphavirus particle contains a nucleocapsid structure that is contained within a host cell-derived lipid bilayer, such as a plasma membrane, in which alphaviral-encoded envelope glycoproteins are embedded. Use of the term "recombinant" when referring to alphavirus particles means that the alphavirus particles that have been generated or modified by molecular genetic manipulation, and is does not refer to wild-type alphaviruses as found in nature. Recombinant alphavirus particles however may be derived from alphaviruses found in nature, when all or a portion of such an aiphavirus is used to construct or otherwise generate a recombinant alphavirus particle.

"Alphavirus packaging cell line" refers to a cell which contains an alphavirus structural protein expression cassette and which produces recombinant alphavirus particles after introduction of an alphavirus vector construct, RNA
vector replicon. eukaryotic layered vector initiation system, or recombinant alphavirus particle.
The parental cell may be of mammalian or non-mammalian origin. Within preferred embodiments, the packaging cell line is stably transformed with the structural protein expression cassette.
"Eukarvotic Layered Vector Initiation System" or "ELVIS" refers to an assembly which is capable of directing the expression of a sequence(s) or gene(s) of interest. The eukaryotic layered vector initiation system should contain a 5' promoter which is capable of initiating in vivo (i.e., within a cell) the synthesis of RNA from cDNA, and a viral vector sequence which is capable of directing its own replication in a eukaryotic cell and also expressing a heterologous sequence. In preferred embodiments, the nucleic acid vector sequence is an alphavirus-derived sequence and is comprised of a 5' sequence which is capable of initiating transcription of an alphavirus RNA (also referred to as 5' CSE, in background), as, well as sequences which, when expressed, code for biologically active alphavirus nonstructural proteins (e.g., nsPl, nsP2, nsP3, nsP4), and an alphavirus RNA polymerase recognition sequence (also referred to as 3' CSE, in background). In addition, the vector sequence may include a viral subgenomic "junction region" promoter, sequences from one or more structural protein genes or portions thereof, extraneous nucleic acid molecule(s) which are of a size sufficient to allow optimal amplification, a heterologous sequence to be expressed, one or more restriction sites for insertion of heterologous sequences, as well as a polyadenylation sequence. The eukaryotic layered vector initiation system may also contain splice recognition sequences, a catalytic ribozyme processing sequence, a nuclear export signal, and a transcription termination sequence.
"Antigen" refers to a molecule containing one or more epitopes (either linear, conformational or both) that will stimulate a host's immune system to make a humoral and/or cellular antigen-specific response. The term is used interchangeably with the term "immunogen." Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL
epitope. will include at least about 7-9 amino acids and a helper T-cell epitope at least about 12-20 amino acids. Normally, an epitope will include between about 7 and amino acids, such as, 9, 10, 12 or 15 amino acids. The term "antigen" denotes both subunit antigens, (i.e., antigens which are separate and discrete from a whole organism with which the antigen is associated in nature), as well as, killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microbes. Antibodies such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, which can mimic an antigen or antigeni,' determinant, are also captured under the definition of antigen as used herein. Similarly, an oligonucleotide or polynucleotide which expresses an antigen or antigenic determinant in vivo, such as in gene therapy and DNA
immunization applications, is also included in the definition of antigen herein.
For purposes of the present invention, antigens can be derived from any of several known viruses, bacteria, parasites and fungi, as described more fully below.
The term also intends any of the various tumor antigens. Furthermore, for purposes of the present invention, an "antigen" refers to a protein or peptide which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens.
An "immune response" to an antigen or composition is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest. For purposes of the present invention. a "humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T-lymphocytes and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells ("CTL"s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes. or the lysis of cells infected with such microbes.
Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of.
nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A "cellular immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T_ cells.
A composition or vaccine that elicits a cellular immune response may serve to sensitize a vertebrate subject by the presentation of antigen in association with MHC molecules at the cell surface. The cell-mediated immune response is directed at, or near, cells presenting antigen at their surface. In addition, antigen-specific T-lymphocytes can be generated to allow for the future protection of an immunized host.
The ability of a particular antigen to stimulate a cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject.
Such assays are well known in the art. See, e.g., Erickson et al.. J. Inimztnol. 151:4189-4199, 1993;
Doe et al., Eur. J. Immunol. 24:2369-2376, 1994. Recent methods of measuring cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells (e.g., by the tetramer technique)(reviewed by McMichael, A.J., and O'Callaghan, C.A., J.
Exp.
Med. 187(9):1367-1371, 1998; Mcheyzer-Williams. M.G. et al., Immunol. Rev.
150:5-21, 1996; Lalvani, A. et al., J. Exp. Med. 186:859-865, 1997).
Thus, an immunological response as used herein may be one which stimulates the production of CTLs, and/or the production or activation of helper T-cells. The antigen of interest may also elicit an antibody-mediated immune response.
Hence, an immunological response may include one or more of the following effects:

the production of antibodies by B-cells; and/or the activation of suppressor T-cells and/or T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays known in the art.
"Immature Dendritic Cells" refers to a population of cells with particular characteristics as outlined below. The criteria for identifying immature dendritic cells, both in cytokine-driven culture and from primary tissues, include their dendritic morphology, motility, phagocytic activity. Immature dendritic cells express surface CD 1 a, moderate expression. of surface MHC and costimulatory molecules (e.g.
CD40, CD80, CD86) and are negative for CD3, CD19, CD20 and CD83 (Banchereau and Steinman, Nature 392, 245-252, 1998). These cells have the capacity to differentiate into mature immunostimulatory antigen presenting cells, both dendritic cells and macrophages, the latter being generated via a CD 14+ cell intermediate (Celia et al. Curr Opin in hnmunol. 9:10-16, 1997). Mature dendritic cells (CD86+, MHC+ and CD83+, CD14-) and macrophages (CD14+, MHC+ c-fms+,CD83-) can present processed antigens to immune cells and are thus referred to as professional antigen-presenting cells (Hart, Blood 90: 3245-3287, 1997).
"Infects human dendritic cells" refers to recombinant alphavirus particles or alphaviruses that efficiently infect or transduce human dendritic cells. In this context, transduction or infection efficiency refers to the ability of a recombinant alphavirus particle or alphavirus to bind to, penetrate and deliver an RNA
vector replicon or alphavirus genome to the cytoplasm of a host cell, thereby allowing replicon mediated expression of a heterologous sequence (e.g., a reporter or antigen), or genome-mediated expression of structural proteins in a host cell. Generally, recombinant alphavirus particles or alphaviruses will efficiently transduce or infect host cells in vitro at 10, 20, 25, or 50 fold over a wild-type alphavirus of the same species at an MOI of 100 or less. Alternatively, transduction or infection efficiency is at least 20, 30. 40, or 50% of cells when performed in vitro at an MOI of 100 or less. Wild-type alphaviruses for comparison, as well as cDNA clones for use in the construction of corresponding replicons and structural protein expression cassettes, can be obtained by using full-length virus clones referenced in the following publications: Sindbis virus -Rice et at., 1987, J. Virol. 61:3809-3819; Semliki Forest virus - Liljestrom et al., 1991, J. Virol.
65:4107-4113; Ross River virus - Kuhn et al., 1991, Virology 182:430-441;
Venezuelan equine encephalitis virus - Davis et at., 1989, Virology 171:189-204.

"Isolated aphaviruses" refers to alphaviruses that are separated from the cells in which they are propagated. Alphaviruses may be further purified by a number of techniques, including for example, plaque purification.

As noted above, the present invention provides compositions and methods suitable for generating an immune response in a warm-blooded animal (e.g., a human, horse, cow, sheep, pig, dog, cat, rat or mouse), comprising the general step of administering to an animal a gene delivery vector (e.g., alphavirus vector particles, ELVIS, DNA), or, protein followed by the step of administering to the same animal the same or similar antigen by a second gene delivery vector (e.g. alphavirus vector particles, ELVIS, or, DNA), or, protein, wherein the first and second methods are different. By administering two separate gene delivery vectors (or a vector and protein), one can boost the immune response of the warm-blooded animal to the desired antigen.

In order to further an understanding of the present invention, described in more detail below are (A) methods for generating alphavirus-based gene delivery vectors and DNA-based gene delivery vectors; and (B) methods of utilizing one or both in a variety of methods.

A. Alphavirus-based gene delivery vectors, and DNA-based -gene delivery vectors A wide variety of alphavirus-based gene delivery vectors may be readily generated using the disclosure provided herein. Representative examples of such vectors include RNA vector replicons, alphavirus vector constructs, and recombinant alphavirus particles. Briefly, sequences encoding wild-type alphaviruses suitable for use in preparing the above-described vectors can be readily obtained from naturally-occurring sources, or from depositories (e.g., the American Type Culture Collection, Rockville. Maryland). In addition, wild-type alphaviruses may be utilized for comparing their ability to infect dendritic cells, macrophages or antigen presenting cells with the alphaviruses and derived vectors of the present invention.
Representative examples of suitable alphaviruses include Aura virus (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou virus (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan virus (ATCC
VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach virus (ATCC
VR-927), Mayaro virus (ATCC VR-66, ATCC VR-1277). Middleburg virus (ATCC
VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu virus (ATCC
VR-371), Pixuna virus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC
VR-373, ATCC VR-1246), Semliki Forest virus (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248; see also CMCC #4640, described below), Tonate virus (ATCC VR-925), Triniti virus (ATCC VR-469), Una virus (ATCC VR-374), Venezuelan equine encephalomyelitis virus (ATCC VR-69, ATCC
VR-923, ATCC VR-1250 ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis virus (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa virus (ATCC VR-926), and Y-62-33 virus (ATCC VR-375).
Representative examples of suitable methods for constructing alphavirus-based vector systems are described in more detail in PCT
Publication No.
WO 97/38087 entitled "Recombinant Alphavirus-Based Vectors With Reduced Inhibition Of Cellular Macromolecular Synthesis" and U.S. Patent Nos.
5,091,309 and 5,217,879, 5,843,723, and 5,789,245.
Within one aspect of the invention, a variety of expression cassettes are provided, which contain the sequences coding for and operably express one or more alphavirus structural polypeptides provided herein. Generally, the expression cassettes fall within one of three categories: 1) a DNA promoter of RNA transcription (e.g., RNA
polymerase II promoter) directly and operably linked to the structural protein open reading frame (ORF), and a transcription terminationipolyadenylation sequence;
2) an alphavirus defective helper RNA transcribed in vitro or in vivo, comprising the ordered elements 5' viral or defective-interfering RNA sequence required in cis for alphaviral replication (also referred to as 5' CSE, in background), viral subgenomic junction region promoter. alphavirus structural protein sequence of the present invention, 3' alphaviral sequence required in cis for replication (also referred to as 3' CSE, in background), and polyadenylate tract; and 3) DNA cassettes comprising the ordered elements of a DNA
promoter of RNA transcription that functions within a eukaryotic cell (e.g., RNA
polymerase II promoter) and is operably linked to a 5' viral or defective-interfering RNA sequence required in cis for alphaviral replication, viral subgenomic junction region promoter, alphavirus structural protein sequence of the present invention, 3' alphaviral sequence required in cis for replication, polyadenylate tract, and transcription termination/polyadenylation sequence. In preferred embodiments, the structural proteins of the present invention are synthesized at high levels by the cassettes only after induction by the RNA vector replicon itself or some other provided stimulus. In further embodiments, the structural protein expression cassettes do not express all alphavirus nonstructural proteins.
Within further aspects of the invention, alphavirus packaging cell lines are provided. In particular, within one aspect of the present invention, alphavirus packaging cell lines are provided wherein the viral structural proteins are supplied in trans from one or more stably transformed expression cassettes, and are able to encapsidate transfected, transduced, or intracellularly produced alphavirus vector RNA
transcripts in the cytoplasm and release functional packaged vector particles through the plasma membrane. In preferred embodiments, the structural proteins necessary for packaging are synthesized at high levels only after induction by the RNA
vector replicon itself or some other provided stimulus, and the transcripts encoding these structural proteins are capable of cytoplasmic amplification in a manner that will allow expression levels sufficient to mimic that of a natural viral infection (WO
97/38087 and U.S. Patent 5,789,245). In other embodiments. the structural proteins may be specifically modified prior to use in alphavirus packaging cell lines (e.g., sequence coding for the nuclear localization signal of VEE capsid protein may be altered by site-directed mutagenesis to prevent this function). Furthermore, in other embodiments, expression of a selectable marker is operably linked to the structural protein expression cassette. Such a linked selectable marker allows efficient generation of stable packaging cell lines.
As provided by the invention, methods for generation (packaging) of recombinant alphavirus vector particles are provided and may be readily accomplished for example, by co-transfection of complementing vector and defective helper (DH) molecules as in vitro transcribed RNA or plasmid DNA, or by co-infection with virus (see Xiong et al., Science 243:1188-1191, 1989, Bredenbeek et al., J Virol.
67:6439-6446, 1993, Dubensky et al., J. Virol 70:508-519, 1996 and U.S. Patents 5,814,482, 5,739,026, 5,766,602, 5,789,245 and 5.792,462. Alternatively, vector particles may be generated by introduction of vector RNA into stable alphavirus packaging cell lines or "PCL" (Polo et al., PNAS 96:4598-4603; U.S. Patent 5,789,245). Briefly, such PCL
and their stably transformed structural protein expression cassettes can be derived using methods described within U.S. Patent 5,789,245, or using novel methods described within this invention. For example, the production of recombinant alphavirus vector particles by PCL may be accomplished following introduction of alphavirus-based vector molecules into the PCL, the vectors being derived from in vitro transcribed RNA, plasmid DNA, or previously obtained recombinant alphavirus particles, incubating the PCL for a under conditions and for a time necessary for vector particle packaging, and harvesting of the packaged vector particles. Packaging cell lines may be used for the serial propagation of alphavirus vector particles following high or low multiplicity of infection.
In addition to the above viral-based vectors, numerous non-viral gene delivery vehicles may likewise be utilized within the context of the present invention.
Representative examples of such gene delivery vehicles include direct delivery of nucleic acid expression vectors or naked DNA alone (see e.g., U.S. Patent Nos.
5,814.482 and 5,580,859), B. Use of gene therapy vectors As noted above. within one aspect of the present invention methods are provided for generating or enhancing an immune response utilizing one or more gene delivery vectors (e.g., alphavirus vectors) and/or recombinant protein in order to prime and boost an immune response. Representative examples of such disease targets include viral infections such as HIV, HBV, HCV, HTLV I, HTLV II, CMV, EBV, HSV, respiratory syncytial virus, HPV, as well as cancer antigens (e.g., melanoma).
More specifically, within one aspect of the present invention, compositions and methods are provided for stimulating an immune response (either humoral or cell-mediated) to a pathogenic agent, such that the pathogenic agent is either killed or inhibited. Representative examples of pathogenic agents include bacteria.
fungi, parasites, viruses and cancer cells.
This approach presents a distinct advantage over others since the antigenic epitopes expressed can be altered by selective cloning of sub-fragments of the gene for the antigen into the alphavirus vector, leading to responses against immunogenic epitopes which may otherwise be overshadowed by immunodominant epitopes. Such an approach may be extended to the expression of a peptide having multiple epitopes, one or more of the epitopes being derived from different proteins.
Further, this aspect of the invention allows efficient stimulation of cytotoxic T
lymphocytes (CTL) directed against antigenic epitopes, and peptide fragments of antigens encoded by sub-fragments of genes, through intracellular synthesis and association of these peptide fragments with MHC Class I molecules. This approach may be utilized to map major immunodominant epitopes for CTL induction.
An immune response may also be achieved by transferring to an appropriate immune cell (such as a T lymphocyte) the gene for the specific T
cell receptor which recognizes the antigen of interest (in the context of an appropriate MHC
molecule if necessary), for an immunoglobulin which recognizes the antigen of interest.
or for a hybrid of the two which provides a CTL response in the absence of the MHC
context. Thus, the gene delivery vehicle cells may be used as an immunostimulant, immunomodulator, or vaccine.

One particularly appealing use for alphavirus vectors is human vaccine and therapeutic application. As described in the detailed description and examples below, the present invention provides compositions and methods to increase the potency of such aiphavirus-based systems, particularly in the area of vaccines. One approach exemplified by the present invention is the targeting of alphavirus vectors directly to antigen presenting cells (e.g., dendritic cells, macrophages). It is known that among the many members of the aiphavirus genus, Venezuelan equine encephalitis (VEE) virus is naturally lymphotropic. while the other members are not (Caley et al., J.
Virol. 71:3031-3038, 1997). Yet it has also been observed that one member of the alphavirus group, VEE (MacDonald et al., J. Virol. 74:914-922), and now SFV
and SIN
(Figure 8) are capable of infecting murine dendritic cells at relatively high efficiency.
Unfortunately, the ability of an alphavirus to efficiently infect murine dendritic cells is not predictive of its ability to efficiently infect human dendritic cells (Figure 8), which would be a goal of vaccine optimization for human use with these vectors. It should also be noted that VEE virus is a significant human pathogen responsible for sporadic epidemic outbreaks of encephalitis, resulting in multiple deaths or permanent central nervous system sequelae in virus infected individuals. Thus, VEE is classified for use under BL-3 level containment, suggesting that its development as a vaccine vector is somewhat dubious.
As illustrated below, alphaviruses. including those considered to be non-lymphotropic alphaviruses (see Caley et al., ibid) may be modified or adapted for efficient infection of human dendritic cells, their genomes then cloned, sequenced, and genetic modifications responsible then used in the construction of vectors and packaging cassettes useful for gene delivery to human dendritic cells. It should also be noted that this method of modification or adaptation to specifically target desired human cell types or populations is not limited to dendritic cells. Rather, the method can be extended to other relevant target cell types (e.g., cancer cells) by using the same process of cycling virus or vector through.primary cultured cells. followed by cloning and construction of vectors and packaging cassettes. The invention also provides additional strategies to enhance the vaccine efficacy of alphavirus-based vectors, by utilizing prime-boost regimes that comprise administering or expressing a desired antigen using two separate and different methods, selected for example from alphavirus vector particles, nucleic acid (e.g., DNA) or recombinant protein, and combined in any variety of order, provided that either the prime or the boost or both is an alphavirus derived vector.
Administration of the vectors and/or recombinant proteins of the present invention to a vaccinee, for example in the prime/boost strategy, may be accomplished by a variety of routes. For example, within one embodiment a warm-blooded animal may be primed by a first method (e.g., alphavirus vector particles. ELVIS, DNA, or recombinant protein) that stimulates an immune response against the desired antigen(s).
A wide variety of inoculation routes may be utilized in this regard, including 'for example, intraocularly, intranasally, sublingually, orally, topically, intravesically, intrathecally, intravaginally, intrarectally topically, intravenously, intraperitoneally, intracranially, intramuscularly, intradermally, or, subcutaneously. Priming by the first method may include multiple administrations of the selected method. Following priming, the same or substantially the same antigen is administered to the same animal via a second method (e.g., alphavirus vector particles, ELVIS, DNA, or recombinant protein) that stimulates an immune response against the desired antigen(s).
Similar to the priming step, wide variety of inoculation routes may be utilized in this regard (see above list). Within certain embodiments. the boosting step may be performed several times, as is best suited to generate a protective or therapeutic immune response.
Within yet other aspects of the present invention, the above-described alphavirus-based vector system (e.g., alphavirus vector particles, RNA vector replicons, eukaryotic layered vector initiation systems and the like) may be used for non-vaccine therapeutics, such as the treatment of coronary artery disease (CAD) and/or peripheral vascular disease (PVD). For example, within one embodiment an alphavirus-based vector such as ELVIS or recombinant alphavirus particle can be administered to a patient to treat or prevent CAD or PVD. In cardiovascular indications.
including CAD
and PVD, alphavirus vectors, both DNA- and particle-based can be used to deliver particular palliatives which stimulate angiogenesis and/or neovascularization.
Included among these palliatives are. for example, fibroblast growth factor (e.g., FGF
1, 2, 4, 5, 18), vascular endothelial growth factor (e.g., VEGF-2, D), endothelial nitric oxide synthetase (e.g., NOS). Delivery of these vectors for therapeutic effect can be accomplished by several alternative routes, including for example intramuscular for PVD, and intracoronary, intramyocardial, or perivascular. for CAD.
Adjuvants may also be used to enhance the effectiveness of the vaccinating compositions used for priming or boosting. Such adjuvants include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (International Publication No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5%
Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model I IOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10%
Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RibiTM adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL +CWS
(DetoxT11t);
(3) saponin adjuvants, such as StimulonT"t (Cambridge Bioscience, Worcester, MA) may be used or particle generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (IL-I, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) oligonucleotides or polymeric molecules encoding immunostimulatory CpG motifs (Davis. H.L., et al., J.
Immunology /60:870-876, 1998; Sato, Y. et al., Science 273:352-354. 1996) or complexes of antigens/oligonucleotides. Polymeric molecules include double and single stranded RNA and DNA, and backbone modifications thereof, for example.

methylphosphonate linkages. Further, such polymeric molecules include alternative polymer backbone structures such as, but not limited to, polyvinyl backbones (Pitha, Biochem Biophts Acta 204:39, 1970a; Pitha, Biopolvmers 9:965, 1970b), and morpholino backbones (Summerton, J., et al., U.S. Patent No. 5,142,047. issued 08/25/92; Summerton, J., et al., U.S. Patent No. 5,185,444 issued 02/09/93). A
variety of other charged and uncharged polynucleotide analogs have been reported.
Numerous backbone modifications are known in the art, including, but not limited to, uncharged linkages (e.g., methyl phosphonates, phosphotri esters, phosphoamidates, and carbamates) and charged linkages (e.g., phosphorothioates and phosphorodithioates)_~;
and (7) other substances that act as immunostimulating agents to enhance the effectiveness of the VLP immune-stimulating (or vaccine) composition. Alum, CpG
oligonucleotides, and MF59 are preferred.
A variety of genes encoding polypeptide antigens can be used in the practice of the present invention. Antigens can be derived from a wide variety of viruses, bacteria, fungi, protozoans and other parasites. For example, the present invention will find use for stimulating an immune response against a wide variety of proteins or peptides from the herpes virus family, including proteins derived from herpes simplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2 gB, gD, gH, VP16 and VP22; antigens derived from varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) including CMV gB and gH; and antigens derived from other human herpesviruses such as HHV6 and HHV7. (See, e.g. Chee et al., Cvtomegaloviruses (J.K. McDougall, ed., Springer-Verlag 1990) pp. 125-169, for a review of the protein coding content of cytomegalovirus; McGeoch et al., J.
Gen. Virol.
69:1531-1574, 1988, for a discussion of the various HSV-l encoded proteins;
U.S.
Patent No. 5,171,568 for a discussion ofHSV-1 and HSV-2 gB and gD proteins and the genes encoding therefor; Baer et al., Nature 310:207-211, 1984. for the identification of protein coding sequences in an EBV genome; and Davison and Scott, J. Gen.
Virol.
67:1759-1816, 1986, for a review of VZV.) Additionally, immune responses to antigens from viruses associated with hepatitis, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV), and hepatitis G virus, can also be stimulated using the constructs of the present invention. By way of example, the HCV genome encodes several viral proteins, including the structural core.
El (also known as E) and E2 (also known as E2/NSI) proteins. as well as nonstructural proteins (e.g., NS3, NS4, NS5), which will find use with the present invention (see, Houghton et al. Heparology 14:381-388, 1991, for a discussion of HCV proteins, including El and E2). The delta-antigen from HDV can also be used (see, e.g., U.S.
Patent No. 5,389,528, for a description of the delta-antigen).
Similarly, influenza virus is another example of a virus for which the present invention will be particularly useful. Specifically, the envelope glycoproteins HA and NA of influenza A are of particular interest for generating an immune response.
Numerous HA subtypes of influenza A have been identified (Kawaoka et al., Virology 179:759-767, 1990; Webster et al. "Antigenic variation among type A influenza viruses," p. 127-168. In: P. Palese and D.W. Kingsbury (ed.), Genetics of influenza viruses. Springer-Verlag, New York).
Other antigens of particular interest to be used in the practice of the present invention include antigens and polypeptides derived therefrom from human papillomavirus (HPV), such as one or more of the various early proteins including E6 and E7: tick-borne encephalitis viruses; and HIV-1 (also known as HTLV-111, LAV, ARV. etc.), including, but not limited to, antigens such as gp120. gp4l, gpl60. Gag and pol from a variety of isolates including, but not limited to, HIV111b, HIVSFõ
HIV-lsF162' HIV-ISF170, HIVLAV, HIVE.,,, HIVMN, HIV-ICM,35,, HIV- las, other HIV-1 strains from diverse subtypes (e.g., subtypes, A through G, and 0), HIV-2 strains and diverse subtypes (e.g., HIV-2L, and HIV-2õc,). See, e.g., Myers. et al., Los Alamos Database, Los Alamos National Laboratory, Los Alamos, New Mexico; Myers, et al., Human Retroviruses and Aids, 1990, Los Alamos, New Mexico: Los Alamos National Laboratory.
Proteins derived from other viruses will also find use in the methods described herein, such as for example, proteins from members of the families Picornaviridae (e.g., polioviruses, etc.); Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae; Coronavindae; Reoviridae; Bimaviridae;
Rhabdoviridae (e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g., influenza virus types A. B and C, etc.); Bunyaviridae; Arenaviridae; Retroviradae, e.g., HTLV-I;
HTLV-II; HIV-1; HIV-2; simian immunodeficiency virus (SIV) among others. See, e.g., Virology, 3rd Edition (W.K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B.N. Fields and D.M. Knipe, eds. 1991; Virology, 3rd Edition (Fields, BN, DM
Knipe, PM Howley, Editors, 1996, Lippincott-Raven, Philadelphia, PA) for a description of these and other viruses.
Particularly preferred bacterial antigens are derived from organisms that cause diphtheria, tetanus, pertussis, meningitis, and other pathogenic states, including, without limitation, antigens derived from Corvnebacterium diphtheriae, Clostridium:
=tetani, Bordetella pertusis, Neisseria meningitides, including serotypes Meningococcus A, B, C, Y and W135 (MenA, B, C, Y and W135), Haemophilus influenza type B
(Hib), and Helicobacter pylori. Examples of parasitic antigens include those derived from organisms causing malaria and Lyme disease.
Furthermore, the methods described herein provide means for treating a variety of malignant cancers. For example, the system of the present invention can be used to enhance both humoral and cell-mediated immune responses to particular proteins specific to a cancer in question, such as an activated oncogene, a fetal antigen, or an activation marker. Tumor antigens include any of the various MAGEs (melanoma associated antigen E), including MAGE 1, 2, 3, 4, etc. (Boon, T. Scientific American (March 1993):82-89); any of the various tyrosinases; MART I (melanoma antigen recognized by T cells), mutant ras; mutant p53; p97 melanoma antigen; CEA
(carcinoembryonic antigen). among others.

C. Deposit Information The following materials have been deposited with the American Type Culture Collection:

Deposit Designation Deposit Date Accession No.
Wild type Sindbis virus CMCC #4639 April 2, 1996 VR-2526 SinDCchiron virus NA April 13, 1999 VR-2643 The above materials were deposited by Chiron Corporation with the American Type Culture Collection (ATCC), under the terms of the Budapest Treaty on the International Recognition of the Deposit' of Microorganisms for purposes of Patent Procedure. The accession number is available from the ATCC at telephone number (301) 881-2600.
These deposits are provided as convenience to those of skill in the art, and are not an admission that a deposit is required. The nucleic acid sequence of these deposits, as well as the amino acid sequence of the polypeptides encoded thereby should be referred to in the event of an error in the sequence described therein. A license may be required to make, use, or sell the deposited materials, and no such license is granted hereby.
The following examples are included to more fully illustrate the present invention. Additionally, these examples provide preferred embodiments of the invention and are not meant to limit the scope thereof. Standard methods for many of the procedures described in the following examples, or suitable alternative procedures, are provided in widely reorganized manuals of molecular biology, such as, for example, "Molecular Cloning," Second Edition (Sambrook et al.. Cold Spring Harbor Laboratory Press, 1987) and "Current Protocols in Molecular Biology" (Ausubel et al., eds. Greene Associates/Wiley Interscience, NY, 1990), as well as in U.S. Patents 6,015,686, 5,814,482, 6,015694, 5,842.723, and 5,789,245, and as such.

EXAMPLES
EXAMPLE I

SELECTION AND CLONING OF ALPHAVIRUS VARIANTS THAT INFECT PRIMARY HUMAN
DENDRITIC CELLS

In order to demonstrate that viruses of the Alphavirus genus, including those previously characterized as non-lymphotropic or previously shown to infect murine dendritic cells, could be modified or adapted to efficiently infect and propagate in human dendritic' cells, Sindbis virus was chosen as a representative example. Other similar alphaviruses, such as Semliki Forest virus, Venezuelan equine encephalitis virus and Ross River virus, also may be readily substituted by one of skill in the art, using the disclosure provided herein. Using a naturally occurring heterogeneous virus sample that was propagated only a very limited number of cycles in common laboratory cell lines, adaptation was performed as outlined in Figure 1. Briefly, the virus was passaged 4 times in primary human dendritic cells obtained from different donors, with intermediate plaque purification in 293 and BHK-21 cells.
The primary human dendritic cells used for virus passage were derived from peripheral blood monocytes as previously described (Bender et al., J
Immunol.
Meth. 196:121, 1996). The buffy coat population of cells was obtained from healthy donors at the Blood Center of the Pacific (San Francisco. CA) or Stanford Medical School Blood Center (Palo Alto, CA). CD14+ monocytes were isolated by negative depletion using Monocyte Isolation Kits and mini-MACS columns (Miltenyi Biotec GmbH), according to the manufacturer's instructions. Dendritic cells were generated from the CD14+ cells by culturing at 0.6x106 per ml in RPMI 1640 medium, supplemented with 10% FCS, 2 mM glutamine, penicillin/streptomycin, 1,000 U/ml rhGM-CSF (Peprotech), and 1,000 U/ml rhIL-4 (Peprotech). Culture medium containing cytokines was replenished every two days. Monocyte-conditioned medium (MCM) was prepared as previously described (Bender et al., ibid) and was added to immature DC cultures at 30% (vol/vol) to induce maturation, either 5 or 6 days following culture initiation, for 3 additional days. Expression of cell surface markers upon MCM-treatment was analyzed by flow cytometry.
Following dendritic cell adaptation, a panel of plaque-purified clonal virus variants was able to grow efficiently in primary human dendritic cells, producing virus titers of greater than 108 PFU/ml, or 1000 infectious virus particles/cell. Each clonal variant formed small plaques on BHK-21 and Vero cells, as compared to the parental virus and other common laboratory Sindbis virus strains. One of the DC-selected plaque-purified viruses was chosen for cDNA cloning, and an agarose plug from the last plaque purification step was incubated in I nil of media (MEM
with 10%
FBS) for 4 hours at 4 C. A 100 ul aliquot of virus eluate then was used to infect 106 BHK-21 cells. After development of CPE the media (5 ml) was harvested. This virus stock produced homogeneous small plaques on both BHK-21 and Vero cells, and the titer was 2x108 PFU/ml. Virus from this seed stock, designated SinDCchiron, was deposited with American Type Culture Collection according to the requirements of the Budapest Treaty.
In addition, a small number of spontaneous large plaque variants were observed sporadically in BHK-21 and Vero cells. The plaque size of these variants was significantly larger than the parental virus and other common laboratory strains of Sindbis virus. Similar to other alphaviruses, this large plaque virus variant was inefficient at infecting primary human dendritic cells and produced very low titers of progeny virus (<l0' PFU/ml). One of the large plaque variants was chosen for cDNA
cloning, and an agarose plug from the last plaque purification step was incubated in I
ml of media (MEM with 10% FBS) for 4 hours at 4 C. A 100 ul aliquot of virus eluate then was used to infect 106 BHK-21 cells- After development of CPE the media (5 ml) was harvested. This virus stock produced homogeneous large plaques on both BHK-and Vero cells. Virus from this seed stock was designated SinChironLP.
Approximately 10' BHK-21 cells were infected with either the dendritic cell adapted virus seed stock or the large plaque virus seed stock at a MOI of I
PFU/cell. At 24 hours post-infection, after development of CPE, total RNA was isolated from the cells using the TRIzol Reagent (GibcoBRL) according to the manufacturer's instructions. After purification, viral RNA was dissolved in nuclease-free water, aliquoted, and stored at -80 C for later use in cDNA cloning (Figure 2A).

Synthesis of cDNA was accomplished by PCR amplification, using the primer sets shown below (Sindbis nucleotide numbering indicated for each primer):

1.1 CCACGAA TTCAGCCAGATGAGTGAGGC 10364-10381 2.1 CCACGAA TTCATATGGGGAAATCATGAGCC 9614-9634 3.1 CCACGAA TTCAAGATTAGCACCTCAGGACC 8878-8899 4.1 CCACGAA TTCGTCCGATCATGGATAACTCC 8294-8315 5.1 CCACGAA TTCACTGCCATGTGGAGGCC 7797-7814 6.1 CCACGAGCTCGCGACATTCAATGTCGAATGC 6426-6446 7.1 CCACGAGCTCGACCTTGGAGCGCAATGTCC 5843-5862 8.1 CCACGAGCTCGACCATGGAAGCAATCCGC 4814-4832 9.1 CCACGAGCTCCACGGAGACAGGCACCGC 4246-4264 10.1 CCACGAGCTCGAACTCTCCCGTAGATTTCC 3407-3427 11.1 CCACGAGCTCCCAGGGGATATCATCCTGAC 2742-2761 12.1 CCACGAGCTCGAACCGCAAACTATACCACATTGC 1976-1999 13.1 CCACGAGCTCGGAGAACGGGTATCGTTCC 1029-1047 14.1 CCACGAGCTCATTGACGGCGTAGTACACAC 1-20 Primer pairs 1-5 were used for cloning of the virus structural genes.
while pairs 6-14 were for the virus nonstructural genes. Oligonucleotides in pairs 1-5 contained additional sequences representing restriction enzyme sites for EcoRl and HindlIl, which are not present in subgenomic RNA of Sindbis virus.
Oligonucleotides 6-14 contained sites for SacI and Xhol, which are not present in the whole genome of previously sequenced strains of Sindbis virus (these sites are underlined).

Each reverse transcription (RT) reaction was performed in a 50 ul volume using the SuperscriptIl enzyme (GibcoBRL), according to the manufacturer's instructions. Reaction mixtures contained the amount of RNA equivalent to 106 cells and 50 pmoles of each primer shown below.

Mixture 1: primersl, 3 and 5 Mixture2: primers 2 and 4 Mixture3: primers 6, 9 and 12 Mixture4: primers 8, 11 and 14 RT reactions were frozen and then used directly for PCR amplification.
PCR reactions were performed using Vent DNA polymerase (NEB) as recommended by the manufacturer. Each 50 ul PCR reaction contained 3 ul of RT mixtures described above and 50 pmoles of primers. A total of 14 reactions were performed (Table 1).

TABLE I

N of fragment Primers N of RT reaction. Length of the fragment (bp.) 1 l and 1.1 1 1128 N of fragment Primers N of RT reaction. Length of the fragment (bp.) 2 2 and 2.1 2 800 3 3 and 3.I 1 789 4 4 and 4.1 2 644 5 and 5.1 1 670 6 6 and 6.1 3 962 7 7 and 7.1 3 666 8 8 and 8.1 4 1107 9 9 and 9.1 3 638 10 and 10.1 3 912 11 11 and 11.1 4 743 12 12and12.1 3 870 13 13 and 13.1 3 1034 14 14 and 14.1 4 1088 PCR reactions for fragments 1-5 were performed using the following conditions: 12 cycles of 95 C for 30 seconds, 56 C for 30 seconds and 74 C for seconds. For fragments 6-14, the number of cycles was changed from 12 to 15. A
small aliquot of each reaction mixture was analyzed by agarose gel electrophoresis to confirm the presence of the fragments of the expected size. The rest was extracted with phenol-chloroform and DNA fragments were precipitated using ethanol.
For cloning, fragments 1-5 were digested with HindIII and EcoRI, and then ligated with plasmid pRS2 (pUC19 with additional restriction sites in polylinker) treated with the same enzymes. Fragments 6-14 were digested with Sacl and Xhol and ligated with the same pRS2 plasmid treated with SacI and XhoI. All recombinant plasmids were transformed into the E. coli XL-1 Blue strain (Stratagene, La Jolla, CA).
In addition, cDNA clones representing the subgenomic promoter region and 3'-end nontranslated regions also were generated using the following primer pairs:

YSINIF
5'-GATTCGGTTACTTCCACAGC
YSINIR
5'-ACTGACGGCTGTGGTCAGTT

5'-GATGTACTTCCGAGGAACTG

'-CCACAAG CTTG AAATGTTAAAAACAAAATTTTGT

Three positive colonies for each transformation were grown in 40 ml of 2xYT media supplemented with ampiciIlin (200 )ig/mI), plasmids were purified using a QIAGEN kit according to the manufacturer's instructions, and the insertions were sequenced. By comparison of sequences from three independent clones, the genome sequence of each virus was determined (Figures 2B and 2C) and compared to published sequences for other Sindbis virus strains. Correct cDNA fragments for each virus (based on consensus of the three independent clones) were designated pI-pl4 correspondingly.
Sequence data demonstrated that the SinDCChiron (also known as DC+) and SinChironLP (also known as LP) strains differed at only a single amino acid residue, throughout their entire genomes. This determinant for efficient infection of human dendritic cells, and in particular immature human DC, was located at E2 glycoprotein residue 160, with the strains containing the amino acids Gly or Glu, respectively. Therefore, substitution of Gly for Glu at E2 160 alone is responsible for conferring the human DC-adapted phenotype. Additional amino acid substitutions, deletions or insertions at, or in close proximity to, this site can be readily generated by standard site-directed mutagenesis protocols, and when inserted into a full-length cDNA
clone described below, also may produce the same human DC adapted growth characteristics.

CONSTRUCTION OF A FULL-LENGTH cDNA CLONE, VECTORS AN*D PACKAGING
CASSETTES FROM HUMAN DENDRITIC CELL ADAPTED ALPHA VIRUSES

The construction of a full-length cDNA clone, replicon vectors, and structural protein expression (packaging) cassettes from a human dendritic cell adapted alphavirus, such as SinDCchiron virus, is readily accomplished by one of skill in the art using the teachings provided below, as well as those previous teachings provided by U.S. Patents 5,814,482, 5,789,245 and 5,843,723. Vector replicon construction from clones of the SinDCchiron virus was accomplished using clones pI-p14 (Example 1) as follows. An Apal-Mscl fragment, containing the promoter for SP6 RNA polymerase and start of the Sindbis virus genomic RNA, was ligated with the Mscl-Xhol fragment of cloned fragment 14 in Apal-XhoI digested plasmid pRS2. The resulting plasmid was named p15. Next, the Sacl-EcoRI fragment of p8, the EcoRI-NsiI fragment of p7 and the NsiI-Xhol fragment of p6 were ligated into Sacl-Xhol digested pRS2. The resulting plasmid was named p16. Next, the Sacl-Muhl fragment of p12, the Muni-Nhel fragment of pl 1 and the NheI XhoI fragment of plO were ligated into SacI XhoI
digested pRS2 plasmid. The resulting plasmid was named p17. The Apal-ApaLI
fragment ofp15 and the ApaLI-XlzoI fragment ofp13 then were ligated into ApaI-Xlrol treated pRS2, resulting in the plasmid named p 18. Next, the ApaI-NsiI
fragment of p 18 and the NsiI-XhoI fragment of p 17 were ligated together in ApaI-XhoI treated pRS2.
The resulting plasmid was named p19. Finally, the ApaI-AvrII fragment of p19.
the AvrII-SaIGI fragment of p9 and the SaIGI-BainHl fragment of p 16 were ligated together into an ApaI-BainHI treated Sindbis replicon vector expressing the GFP
reporter (see Dubensky et al., J. Virol. 70:508-519, 1996, and U.S. Patent 5,843,723). The resulting newly constructed replicon vector expressing GFP reporter was designated SINCR-GFP
(also known as DCSP6SINgfp).
Construction of a defective helper-based packaging cassette from clones of the SinDCchiron virus may be accomplished as follows. A BamHI-SacII
fragment containing the Sindbis virus subgenomic promoter and 5' subgenomic NTR. from the previously described DH-BB helper plasmid (Bredenbeek et al., J. Virol.
67:6439-6446, 1993), a SacII-Nrul fragment from clone p5 and a Nrul-HinaUl fragment from p4 are cloned together into BamHI-HindIII digested pRS2. The resulting plasmid is named p20. Next, the EcoRI-BspHI fragment of clone p3, the BspHI-SpII fragment of clone p2 and the Spll-NsiI fragment of clone pl are cloned into EcoRl-HindIll digested pRS2.
The resulting plasmid is named p21. Finally, the BarHI-Bsu36I fragment of p20 and the Bsu36I-NsiI fragment of p2I are cloned into BarHI-NsiI digested DH-BB
helper plasmid, resulting in the final packaging construct named DCSP6SINdh.

Other variations of defective helper based structural protein expression cassettes (packaging constructs) can be readily constructed. These include cassettes that express the alphavirus structurdl protein genes in a "split" configuration, as well as RNA polymerase II based constructs for use in the derivation of stable packaging cell lines (see U.S. Patents 5,789,245, 6,015,686, 6,015,694, 5,842,723, and 5,814,482). For example, SP6-based defective helpers were constructed to contain only the Sindbis virus envelope glycoprotein genes with a Ross River virus translation enhancer element.
Construction of such plasmids was performed stepwise as follows.
The EcoRI-Bsu36 fragment from 4.4.2 and the Bsu36-HindIII fragment from 1.3.2 were cloned into EcoRl-HindIIl digested pRS2 (designated c1201).
The EcoRI-Bsu36 fragment from 4.4.2 and the Bsu36-HindII1 fragment from 4.3.2 were cloned into EcoRI-HindIII digested pRS2 (designated c1202). The EcoRI-SpII
fragment from 4.2.2 and the SpII-HindIIl fragment from 1.1.2 were cloned into EcoRl-Hindlll digested pRS2 (designated c1203). The EcoRt-BspHI fragment from cl 201 and the BspHI-HindIIl fragment from c1203 were cloned into EcoRI-HindIll digested pRS2 (designated c1221). The EcoRI-BspHI fragment from cl 202 and the BspHI-HindIII
fragment from c1203 were cloned into EcoRl-HindIII digested pRS2 (c1222). The Stul-NsiI fragment of 6221 and the BspHI-Stul fragment of tRNABB/Cdel3rrv (Frolov et al, 1997, J. Virol. 71:2819-2829) were cloned into BspHI-NsiI digested tRNABB/Cdel3rrv (designated c1231). The Stul-NsiI fragment of c1222 and the BspHI-Stul fragment of tRNABB/Cdel3rrv were cloned into BspHI-NsiI digested tRNABB/Cdel3rrv (designated c1232).

When used to package alphavirus vector replicons, the glycoprotein defective helper clone 232 (tRNABB/Cdel3rrvDC) will produce vector particles that efficiently infect human dendritic cells, particularly the immature population of DC, while the glycoprotein defective helper clone 231 (tRNABB/Cdel3rrvNDC) will produce vectors that do not efficiently infect human dendritic cells.
Alphavirus vector particles for use in testing human dendritic cell tropism, as well as for use in vivo (e.g., vaccine administration), may be generated using a variety of previously described methodologies. These methods include for example, co-transfection of in vitro transcribed replicon vector and defective helper packaging RNAs. co-transfection of plasmid DNA-based vector and packaging cassettes, and introduction of vector replicons into stable packaging cell lines (see for example, Dubensky et al., J. Virol. 70:508-519, 1996; Bredenbeek et al., J. Virol.
67:6439-6446, 1993; and U.S. Patents 5,843,723 and 5,789,245).
As shown if Figures 3, 4, 5 and 8 Sindbis-GFP replicon vector that was packaged in the structural proteins from SinDCchiron virus acquired the ability to efficiently infect primary human dendritic cells, particularly immature dendritic cells.
In these experiments, FACS analysis was performed as follows. Developing dendritic cells were harvested from culture, washed in PBS/1%FCS and enumerated by trypan blue exclusion staining. Non-specific binding was blocked by incubation with human Ig, and cells were incubated with propidium iodide to enable subsequent analysis of viable cells. Cells (1-10x 10 ) were stained at 4 C for 30 minutes with flourochrome-conjugated antibodies to dissect the cell population susceptible to infection.
The following antibodies (and their cellular specificities) were used: CDIa-PE
(pan-dendritic cell), CD86-PE (antigen presenting cells: dendritic cells, macrophages, B
cells). CD83-PE (mature dendritic cells), HLA-ABC-PE (many cell types), HLA-DR-PE (antigen presenting cells: dendritic cells, macrophages, B cells), CD3 (T
cells), CD5 (T cells and B cells), CD 16 (NK cells), CD I I c-PE (dendritic cells and macrophages) and CD14-PE (monocvtes). All antibodies were obtained from Becton Dickinson.
Isotype matched controls were used to establish quadrant positions. Analysis was performed on a FACScan (Becton Dickinson), and the data acquired to a Macintosh 7100 computer. CellQuest 3.1 software was used to analyze and display data.

We found that immature human dendritic cells differentiated from monocytes for 3-5 days with IL4 and GMCSF were highly susceptible to infection with the DC-tropic Sindbis vector particles expressing GFP_ The phenotype of the susceptible cell population, based on cell surface markers was CDla'CD83-CD14"CD3"
CD86'HLA-DR-HLA-ABC'CDllc-. Adherent macrophages were differentiated from monocytes with MCSF (50U/ml; R&D Systems, Minneapolis, MN) for 6 days in 12-well Costar plates at 1.5 x 106 cells Iml_ Adherent macrophages (HLA-DR') derived from these cultures were infected with particular Sindbis variant particles.
Macrophages developed in this system are known in the field to be closely related to dendritic cells, and can function as dendritic cell precursors. Purified CD3` T cells were not infected at equivalent MOIs, and a subset of dendritic cell precursors (HLA-DR-CD14d'"'CD3-CD16-CDlaCD5 were infected when heterogeneous PBMC cultures were exposed to SIN replicon particles. Taken together, dendritic cells, and the macrophage and PBMC
precursor populations were susceptible to infection with Sindbis particles, and T cells, B cells and NK cells were refractory to infection.
When similar studies were performed with mouse derived dendritic cells, it was demonstrated that the structural proteins from wild-type alphaviruses (for example, Sindbis virus, Semliki Forest virus) allow for efficient vector particle infection of mouse DC, but not human DC (see Figure 8, SFV or SIN LP). Only by using modified or adapted structural proteins can these vector particles efficiently infect human dendritic cells (see SIN DC+).
Infection of human dendritic cells was receptor mediated and could be inhibited by an anti-virion rabbit polyclonal antibody. For example, GFP-expressing replicon particles packaged with the SinDCchiron structural proteins (2 x 10') were pre-incubated for 30 minutes with either anti-virion antibody or control rabbit polyclonai antibody recognizing mouse Ig (from Sigma, Cat # M-6024) at 1:100 dilution in PBS.
Immature human dendritic cells (2 x I0'; Day 3) were infected for 1 hour and analyzed 24 hours later for GFP expression. Anti-virion antibody blocked infection of dendritic cells by SinDCchiron particles. whereas non-specific antibody had no effect.

We next wished to determine whether alphavirus vector particles selected for human DC-tropism could be used as a method to induce maturation and activation in dendritic cells that have been transduced with the vector, either in vitro or in vivo. As shown in Figure 9. human DC transduced in vitro with SIN-GFP
vector particles, show significant up-regulation of the cell surface markers CD80, CD86, CD83, and MHC class II, which are known markers for maturation and activation.
In addition, similar results were observed in vivo, for a mouse model. Following peripheral inoculation with SIN-GFP vector particles, the CDI Ic dendritic cells positive for GFP expression, were isolated from the draining lymph nodes and shown to have significant up-regulation of maturation and activation markers (e.g., MHC II).
To further demonstrate the ability of DC-tropic Sindbis-GFP particles to infect dendritic cells in vivo, we inoculated groups of 4 female Balb/C mice aged 8-10 weeks intradermally in the ear. The left ear was painted with 25u1 rhodamine B
isothiocyanate (2% dissolved in acetone/dibutylpthalate) prior to injection with 25u1 SIN-GFP particles (1.75 x 10' total particles in formulation buffer). The right ear was not painted with rhodmaine and was injected with SIN-GFP particles (1.75 x 10') alone.
Control groups received formulation buffer alone, and the left ear of each animal was painted with rhodamine. Two animals from each group were anesthetized and exsanguinated at 24 hours and 48 hours post injection, and the ears and draining mandibular node were harvested immediately into 1 % paraformaldehyde. The tissues were fixed for 72 hours at 4 C in the dark, before embedding in paraffin.
Tissue sections (5um) were prepared with a cryostat onto glass microscope slides and analyzed quantitatively with a laser scanning cytometer (Compucyte, Cambridge, MA), or photo-documented with a fluorescent microscope (Zeiss) linked to a CCD camera (Figures 6 and 7). The presence of cells with GFP and rhodamine in the draining lymph nodes provides strong evidence that migratory dendritic cells are infected with sindbis particles in the skin, and traffic to the node. The cells exhibit dendritic morphology and have numerous processes that contact neighboring cells in the node. No GFP
expressing cells were detected in the control groups.
To demonstrate the utility of human DC-adapted alphavirus vectors as a "tool" in the development of in vitro assays for antigen presentation and stimulation of immune cells (e.g., class I restricted T cell responses), we utilized as a representative example, a murine T cell hybridoma assay (Figure 10). The murine T cell hybridoma 12.2 was generated by fusion of splenocytes from HIV-gag-immunized CB6FI mice with the BWZ.36 fusion partner, followed by cloning and selection based on IL-production in response to APCs loaded with HIV-derived peptides. The 12.2 T
cell hybridoma specifically recognizes the peptide sequence AMQMLKETI (p7g) of the HIV-1 SF2 p24gag protein in .the context of H-2Kd. The 12.2 T hybridoma produces IL-2 in a dose-responsive manner upon co-culture with p7g peptide-loaded H-2Kd DC
and is unresponsive to DC loaded with another gag-derived peptide SQVTNPANI
(gagb). The p7g gag-specific T cell hybridoma 12.2 was plated at 106 cells/ml, cells/well in 96-well, U-bottom microtiter plates. Varying numbers of DC were added to wells for a total volume of 2001iI. As a positive control for DC function, DC from each condition were assayed in the presence of I ng/ml p7g peptide, as well as in media alone. Negative control wells containing DC or the T cell hybridoma alone were also included in each experiment and reliably yielded <20 pg/ml IL2. Each experimental condition was assayed in duplicate. After 24 hr co-culture at 37 C, supernatants were removed and assayed for IL-2 production by ELISA according to manufacturer's instructions (Endogen, Woburn, MA). As a permanent source of homogeneous dendritic cell adapted virus, full-length cDNA clones, from which infectious SinDCchiron virus genomic RNA may be transcribed in vitro. may be constructed by using the DCSP6S1Ngfp vector as starting material. Specifically, the .opal-Sa1GI and Sa1GI-BamHI fragments from DCSP6SINgfp vector, plus the BamHI-X7zoI from the DCSP6SINdh packaging construct are cloned into pRS2 that has been digested with Apa1 and A7ioI. The resulting construct is designated DCSP6SINgen. Infectious virus seed stocks may be generated from this plasmid by linearization of the plasmid, transcription in vitro, and transfection of BHK-21 cells as extensively documented in previous publications (see for example Rice et al., J. Virol. 61:3809-3819, 1987;
Dubensky et al., J. i"irol. 70:508-519, 1996; Bredenbeek et al., J. Virol.
67:6439-6446.
1993; and U.S. Patent 5,843,723).

PRIME-BOOST VACCINE STRATEGIES FOR ALPHA VIRUS VECTORS

In order to be optimally efficacious, a vaccine for a given pathogenic agent (e.g., infectious disease agents such as viruses, bacteria, fungi, parasites) must stimulate a robust and broad-based antigen-specific immune response. The vaccinated individual will be resistant to development of sequelae characteristic of the infectious organism upon subsequent challenge, as a result of stimulation of residing memory cells and maturation into antigen-specific effector cells, which facilitate clearing of the offending infectious agent. Similarly, a therapeutic vaccine for a given infectious disease must also stimulate a robust and broad-based antigen-specific immune response, to clear or to diminish the extent of infectious disease.
Generally, vaccines are given in greater than one, and often in many, doses. The rationale of such an immunization strategy is to "boost" the "prime"
response. resulting in a more durable immune response that is characterized by an increased capacity of an individual to resist challenge of an infectious organism. This increased resistance to challenge is due to a larger number of residing immune memory cells, often with specificity for a broader range of antigenic epitopes, corresponding to the infectious organism.
A broad-based antigen-specific immune response results when vaccination elicits both T-helper cell I (Thl) and T-helper cell 2 (Th2) responses.
Proliferation of Thl and Th2 cells are distinguished by their pattern of cytokine secretion, the Thl subset corresponding to interleukin 2 (IL-2), and IFN-y, and the Th2 subset corresponding to IL-4, IL-5, IL-6. and IL-10. Antigens expressed as a result of vaccination are presented to the immune system via dendritic cells (DCs), so-called "professional antigen presenting cells." DCs initiate and modulate the immune response. In particular, DCs are potent stimulators of B and T lymphocytes (for review, see Banchereau and Steinman, Nature 392:245-252. 1998). Stimulation of the T
and B
lymphocyte antigen-specific effectors occurs by presentation by the dendritic antigen presentation cells, in which processed antigen is displayed in conjunction with major histocompatibility complex (MHC) molecules of two alternative types (MHC class 1, or MHC class II), to T lymphocytes, via the T-cell antigen receptor. Antigen presentation via MHC class I results in a cellular CD8+ cell (cytotoxic T cell, CTL) response, whereas antigen presentation via MHC class II results in stimulation of CD4+
cells, and subsequently B lymphocytes, resulting in a humoral (antibody, Ab) response.
Thus, the rationale of a "prime"-"boost" vaccination strategy is to elicit a broad and durable Thl and Th2 antigen-specific immune response.
Traditional vaccination prime-boost strategies have used only a single modality for both the prime and boost steps, and may not, for particular infectious diseases, elicit a broad Th1/Th2 antigen-specific immune response. Thus, within the scope of the prevention invention, several "mixed" modalities of prime-boost regimes are disclosed wherein a variety of vaccinating compositions may be administered either as a "prime" or "boost", in conjunction- with an alphavirus replicon vector (e.g., as recombinant particles, DNA or RNA) encoding a designated antigen. Such a prime-boost immunization strategy incorporating alphavirus vector replicons will elicit a much more durable and broad-based Thl/Th2 antigen-specific immune response in the vaccinated individual. The combined prime-boost immunization modalities may include, for example, plasmid DNA (formulated or non-formulated), ELVIS
vector, recombinant alphavirus particles, other non-aiphaviral vectors and recombinant protein.
Any of these vaccine modalities, including the alphavirus vector replicons encoding a designated antigen, may be given with an adjuvant. Representative examples of adjuvants that can be utilized are MF59, poly-D-galactoside, alum, CpG
oligonucleotides, or mono-phosphoro lipid.
To demonstrate the utility of prime-boost regimes according to the present invention, HIV gag was chosen as an example antigen. HIV gag was expressed by plasmid DNA vectors, ELVIS vector and alphavirus vector particles, and also produced as a recombinant protein. Other antigens from HIV, as well as antigens from other pathogenic agents (for example HCV), similarly may be used by one of skill in the art based on the teachings of the present invention.

A. Construction of HTV gag and envelope expression vectors The HIV gag coding sequence was selected from the HIV-ISF2 strain (Sanchez-Pescador, R., et al., Science 227(4686):484-492, 1985; Luciw, P.A., et al., U.S. Patent No. 5,156,949. issued October 20, 1992;
Luciw, P.A., et al., U.S. Patent No. 5,688.688, November 18, 1997). These sequences have been used directly or first manipulated to maximize expression of their gene products. For maximization of expression, the HIV-1 codon usage pattern was modified so that the resulting nucleic acid coding sequence was comparable to codon usage found in highly expressed human genes. The HIV codon usage reflects a high content of the nucleotides A or T as third base of the codon-triplet. The effect of the HIV-1 codon usage is a high AT content in the DNA sequence that could result in a decreased translation ability and instability of the mRNA. In comparison, highly expressed human codons prefer the nucleotides G or C as the third base. The gag coding sequence therefore was modified to be comparable to codon usage found in highly expressed human genes.
The DNA fragment for gag was cloned into the eukaryotic expression vector pCMVKm2, derived from pCMV6a (Chapman et al., Nuc. Acids Res. 19:3979-3986, 1991) and comprising a kanamycin selectable marker, a CoIEl origin of replication, a CMV promoter enhancer and Intron A, followed by cloning sites for insertion of sequences, followed by a polyadenylation signal derived from bovine growth hormone. The gag sequence-containing vector was designated pCMVKm2.GagMod.SF2. This plasmid was deposited January 18, 1999, with the Chiron Corporation Master Culture Collection. Emeryville, CA, 94662-8097, and with the American Type Culture Collection. 10801 University Boulevard, Manassas. VA
201 1 0-2209.

The DNA fragment encoding HIV gag was then cloned into an alphavirus plasmid DNA vector (ELVIS), and replicon vectors (SINBV and SINCR) to be used for the generation of recombinant alphavirus particles. Specifically, a construct for in vitro transcription of Sindbis virus RNA vector replicons (pRSIN-luc;
Dubensky et al.. J Virol. 10:508-519, 1996) was modified to contain a Pmel site for plasmid linearization and a polylinker for insertion of heterologous genes. First, a polylinker was generated using two oligonucleotides that contain the sites Xhol, Pmll.
Apal, Narl, Xbal, and NotI.

XPANXNF: 5'GCA CGT GGG CCC GGC GCC TCT AGA GC
XPANXNR: 5'GCT CTA GAG GCG CCG GGC CCA CGT GC

The plasmid pRSIN-luc (Dubensky et al., supra) then was digested with Xhol and Notl to remove the luciferase gene insert, blunt-ended using Klenow and dNTPs, and ligated with the oligonucleotides that were annealed to each other.
The resulting construct was digested with Notl and Sacl to remove the minimal Sindbis 3'-end sequence and A,o tract, and ligated with an approximately 0.4 kbp fragment from pKSSINI-BV (WO 97/38087), obtained by digestion with Nod and Sacl. The fragment contained the complete Sindbis virus 3'-end, an A,,, tract and a PmeI site for linearization. This replicon vector construct was designated SINBVE. The other replicon vector, SINCR, was described previously in example 2.
The HIV gag coding sequence was obtained from the parental pCMVKm2.GagMod.SF2 plasmid by digestion with EcoRI, blunt-ending with Klenow and dNTPs, purification with GeneCleanlI. and digestion with Sail. The HIV gag-coding fragment then was ligated into the SINBVE vector that had been digested with Xhol and Pm/I. The resulting vector was designated SINBV-gag. In parallel, the same HIV gag fragment was inserted into the new replicon of the present invention, by ligating with SINCR-GFP vector that had been digested with Noll and blunt-ended.
followed by digestion with XhoI, to remove the GFP reporter gene insert- This vector was designated SINCR-gag. Vector RNA replicons were packaged into recombinant aiphavirus particles by using previously described methods (see for example Dubensky, et al., J Pirol. 70:508-519, 1996; Polo et al., PNAS 96:4598-4603; and U.S.
Patents 5,789,245, 5,842,723, and 6,015,694).

The construction of a new SIN vector replicon utilizing the nonstructural gene sequences obtained from SinDCchiron virus (ATCC# VR-2643) or SinChironLP
virus, described in Figures 2B and 2C, resulted in a replicon that had superior properties as compared to a replicon derived from wild-type SIN virus (e.g., ATCC# VR-2526).
These properties include, but are not limited to, enhanced expression in human dendritic cells, increased packaging into recombinant alphavirus particles at levels as much as 10-fold over wild-type replicons, and the ability to induce a more robust immune response following immunization of animals. For example, Figure I 1 compares the induction of HIV gag specific CD8 T cells in mice following immunization with DC-tropic vector particles containing either the new SIN replicon (SINCR) or a previously described wild-type SIN replicon (SINBV). At doses of either 105 or 10' IU, the SINCR
replicon that contains nonstructural gene sequences from SinDCchiron virus and SinChironLP
virus is clearly more potent at immune induction.
For construction of an alphavirus DNA-based vector (ELVIS) expressing HIV gag, plasmid pDCMVSIN-0-gal (Dubensky et al., J Virol. 70:508-519, 1996) was digested with Sall and Xbal, to remove the beta-galactosidase gene insert, and the HIV
gag gene was inserted after digestion and purification of the fragment from SINBV-gag.
The resulting construct was designated pDSFN-Gag. Similarly, constructs expressing HIV envelope (env) protein were made using the same replicon and ELVIS vector backbones. A variety of envelope sequences have been used for these constructions and the parental plasmids used for isolation of the env genes (e.g., pCMVgpl60.modUS4, pCMVgpl40.mut7.modSF162, pCMVgpl40.mut8.modSF162) have been deposited with CMCC and ATCC.
Recombinant gag protein was obtained using a baculovirus expression system. A baculovirus shuttle vector containing the synthetic HIV gag sequence was constructed as follows. The synthetic HIV p55 gag expression cassette (described above) was digested with restriction enzyme Sail followed by incubation with polymerase. The resulting fragment was isolated and then digested with BaniHl.
The shuttle vector pAcCl3 (Munemitsu S., et al., Mol Cell Biol. 10(11):5977-5982, 1990) was linearized by digestion with EcoRV, followed by incubation with T4-DNA
polymerase. The linearized vector was digested with BamHI, treated with alkaline phosphatase, and the fragment isolated from an agarose gel. The isolated 1.5 kb fragment was ligated with the prepared pAcC13 vector, resulting in the clone designated pAcC 13-Modif.p55gag.

Generation of the recombinant baculovirus was achieved by co-transfecting 2 pg of the HIV p55 gag shuttle vector with 0.5 gg of linearized.
Autographa californica baculovirus (AcNPV) wild-type viral DNA into Spodoptera frugiperda (Sf9) cells (Kitts, P.A., Ayres M.D., and Possee' R.D., Nucleic Acids Res.
18:5667-5672, 1990). The isolation of recombinant virus expressing HIV p55 Gag was performed according to standard techniques (O'Reilly, D.R., L.K. Miller, and V. A.
Luckow, Baculovirus Expression Vector: A Laboratory Manual, W.H. Freeman and Company, New York, 1992).
Expression of the HIV p55 Gag was achieved using a 500 ml suspension culture of Sf9 cells grown in serum-free medium (Maiorella, et al, BiolTechnology 6:1506-1510, 1988) that had been infected with the HIV p55 Gag recombinant baculovirus at a multiplicity of infection (MOI) of 10. Forty-eight hours post-infection, the supernatant was separated by centrifugation and filtered through a 0.2 m filter.
Aliquots of the supernatant were then transferred to PolyclearTM (Beckman Instruments, Palo Alto. CA) ultracentrifuge tubes, underlaid with 20% (wt/wt) sucrose, and subjected to 2 hours centrifugation at 24,00 rpm using a Beckman SW28 rotor.
The resulting pellet was suspended in Tris buffer (20 mM Tris HCI, pH
7.5, 250 mM NaCl, and 2.5 mM EDTA), layered onto a 20-60% (wt/wt) sucrose gradient, and subjected to 2 hours centrifugation at 40,000 rpm using a Beckman SW41 ti rotor. The gradient was then fractionated starting at the top (20%
sucrose) of the gradient into approximately twelve 0.75 ml aliquots. A sample of each fraction was electrophoresed on 8-16% SDS polyacrylamide gels and the resulting bands were visualized after commassie staining. Additional aliquots were subjected to refractive index analysis.
The results indicated that the p55 gag VLP banded at a sucrose density of range of 1.15 - 1.19 g/ml with the peak at approximately 1.17 g/m!. The peak fractions were pooled and concentrated by a second 20% sucrose pelleting. The resulting pellet was suspended in I nil of Tris buffer (described above). The total protein yield as estimated by Bicimchrominic Acid (BCA) (Pierce Chemical, Rockford, IL).
Recombinant HIV envelope proteins were expressed from stable mammalian cell lines, rather than from baculovirus systems. Chinese hamster ovary (CHO) cells were transfected with plasmid DNA encoding the synthetic HIV-1 env proteins using Mirus TranslT-LT1 polyamine transfection reagent (Pan Vera) according to the manufacturers instructions and incubated for 96 hours. After 96 hours, media was changed to selective media (F12 special with 250 1g/ml G418) and cells were split 1:5 and incubated for an additional 48 hours. Media was changed every 5-7 days until colonies started forming at which time the colonies were picked, plated into 96 well plates and screened by gp120 Capture ELISA. Positive clones were expanded in well plates and screened several times for Env protein production by Capture ELISA, as described above. After reaching confluency in 24 well plates, positive clones were expanded to T25 flasks (Corning, Coming, NY). These were screened several times after confluency and positive clones were expanded to T75 flasks.
Positive T75 clones were frozen in LN2 and the highest expressing clones amplified with 0-5 pM methotrexate (MTX) at several concentrations and plated in 100mm culture dishes. Plates were screened for colony formation and all positive closed were again expanded as described above. Clones were expanded an amplified and screened at each step by gp120 capture ELISA_ Positive clones were frozen at each methotrexate level. Highest producing clones were grown in perfusion bioreactors (3L, 100L) for expansion and adaptation to low serum suspension culture conditions for scale-up to larger bioreactors.

The following table shows capture ELISA data from CHO cells stably transfected with plasmid vectors carrying a cassette encoding synthetic HIV-SF162 Env polypeptides (e.g.. mutated cleavage sites, modified codon usage and/or deleted hypervariable regions). Thus, stably transfected CHO cell lines which express Env polypeptides (e.g., gp120, gp140-monomeric, and gp140-oligomenic) have been produced.

CHO Cell Lines Expression Level of SF 162 Envelope Constructs Constructs CHO Clone # MTX f Expression Level*
Level (ng/ml) gp 120.modSF 162 1 0 755-2705 gpl40.modSF162 1 20 nM 180-350 gp140.mut. 1 20 nM 164-451 modSF162 2 20 nM 188-487 3 20 nM 233-804 gpl20.modSF162.de 1 800nM 528-1560 IV2 2 800nM 487-1878 3 800nM 589-1212 gpl40.modSF162.de I 800nM 300-600 1V2 2 800nM 200-400 3 800nM 200-500 CHO Cell Lines Expression Level of SF162 Envelope Constructs Constructs CHO Clone 9 MTX Expression Level*
Level (ng/ml) gp l40.mut. I 800nM 300-700 modSF162.de1V2 2 400nM 1161 3 800nM 400-600 4 400nM 1600-2176 All samples measured at T-75 flask stage unless otherwise indicated The results presented above demonstrate the ability of the constructs of the present invention to provide expression of Env polypeptides in CHO cells.
Production of polypeptides using CHO cells provides (i) correct glycosylation patterns and protein conformation (as determined by binding to panel of MAbs); (ii) correct binding to CD4 receptor molecules; (iii) absence of non-mammalian cell contaminants (e.g., insect viruses and/or cells); and (iv) ease of purification.

B. Demonstration of enhancement by prime-boost vaccination regimes To demonstrate the ability to improve the immunogenicity of vaccines using a prime-boost regime that takes advantage of alphavirus vectors, a variety of different combinations may be used. For example, alphavirus vector particles may be administered as a prime followed by a recombinant protein or DNA vector boost.
Alternatively, a plasmid DNA vector or ELVIS vector may be administered as a prime followed by an alphavir-us vector particle boost. Also, an ELVIS vector prime may be administered with a recombinant protein or non-alphavirus (e.g., poxvirus) vector boost.
It should be evident from the disclosure provided herein that a large number of different combinations of the above prime-boost elements may be interchanged, and that vectors encoding a variety of antigens may be similarly administered, using a variety of immunization routes.

To illustrate the benefits of such prime-boost approaches, animal studies were performed using the SIN-BV-gag vector particles and pCMVKm2.GagMod.SF2 plasmid from above. These studies utilized a highly quantitative vaccinia challenge model to clearly elucidate CTL response differences in immunized animals. Mice were immunized intramuscularly in groups of three mice per sample, with DNA or alphavirus vector particles, in a 100 ul volume. Four weeks later, the mice were boosted with either plasmid DNA or vector particles (same or opposite vaccine compositions as initial priming immunization). Four weeks after boosting, the animals were then challenged intraperitoneally with 10' PFU of recombinant vaccinia virus expressing HIV gag. Five days later:, direct spleen cell CTL assays were performed against gag peptide-pulsed, 51Cr-labeled target cells. As shown in Figure 12, a DNA prime -alphavirus vector particle boost regime resulted in greater immune induction than DNA
alone or vector particles alone.
It should be appreciated, that the order of vaccine components delivered (e.g., alphavirus vector particles, DNA, ELVIS, recombinant protein), as well as the timing, dose and route of immunization, may be changed to include numerous possible combinations from the above. For example, the interval between prime and boost immunizations may range from no interval (simultaneous administration), to one or more days, preferably at least 14 days, and more preferably at least 28 days.
In addition, the route of administration is not limited to intramuscular delivery; rather, other routes (for example, intradermal, intravenous, subcutaneous, intranasal, and oral) also may be utilized.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the scope of the invention Accordingly, the invention is not limited except as by the appended claims.

Claims (3)

1. An alphavirus vector construct, comprising:
(a) a 5' promoter which initiates synthesis of viral RNA in vitro from cDNA;
(b) a 5' sequence which initiates transcription of alphavirus RNA;
(c) a nucleic acid molecule which operably encodes all four alphaviral nonstructural proteins;
(d) an alphavirus RNA polymerase recognition sequence; and (e) a 3' polyadenylate tract, wherein said nucleic acid sequence which operably encodes all four alphavirual nonstructural proteins contains a mutation in at least one nonstructural protein in nsP1 residues 346, 441, 473, nsP2 residues 438, 622, 634, 715, nsP3 residues 417, 456, 505, or nsP4 residue 266, as compared to wild-type.

2. A eukaryotic layered vector initiation system, comprising a 5' promoter capable of initiating in vivo the 5' synthesis of alphavirus RNA from cDNA a sequence which initiates transcription of alphavirus RNA following the 5' promoter a nucleic acid molecule which operably encodes all four alphaviral nonstructural proteins, an alphavirus RNA polymerase recognitition sequence, and a 3' polyadenylate tract, wherein said nucleic acid sequence which operably encodes all four alphaviral nonstructural proteins contains a mutation in at least one nonstructural protein in nsP1 residues 346, 441, 473, nsP2 residues 438, 622, 634, 715, nsP3 residues 417, 456, 505, or nsP4 residue 266, as compared to wild-type.

3. An alphavirus RNA vector replicon capable of translation in a eukaryotic system, comprising a 5' sequence which initiates transcription of alphavirus RNA, a nucleic acid molecule which operably encodes all four alphaviral nonstructural proteins, an alphavirus RNA polymerase recognition sequence and a 3' polyadenylate tract, wherein said nucleic acid sequence which operably encodes all four alphaviral nonstructural proteins contains a mutation in at least one nonstructural protein in nsP1 residues 346, 441, 473, nsP2 residues 438, 622, 634, 715, nsP3 residues 417, 456, 505, or nsP4 residue 266, as compared to wild-type.